POS1049 INTERLEUKIN 40 IS INCREASED IN THE SERUM OF INDIVIDUALS AT RISK OF RHEUMATOID ARTHRITIS AND STIMULATES AN INFLAMMATORY RESPONSE IN MONONUCLEAR CELLS VIA NFKB
Authors: A. Navrátilová, K. Prajzlerová, N. Petrovská, K. Pavelka, J. Vencovský, L. Šenolt, M. Filková, L. Andres Cerezo
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Interleukin 40 (IL-40) is a new cytokine implicated in the pathogenesis of several autoimmune disorders including rheumatoid arthritis (RA). Recently, we found an association of IL-40 with the early stages of RA and demonstrated the pro-inflammatory potential of IL-40 on neutrophils. <h3>Objectives</h3> As the period preceding clinically apparent RA has not yet been fully described with respect to clinical biomarkers, we aimed to investigate IL-40 in individuals who are at risk of RA and to assess its functional mechanism in immune regulation in peripheral blood mononuclear cells (PBMCs). <h3>Methods</h3> IL-40 was determined in the serum of individuals at risk of RA (defined as patients with arthralgia with no clinical arthritis at baseline who are either carriers of anti-citrullinated protein antibodies, ACPA, or meeting the EULAR definition of clinically suspect arthralgia) at baseline (n=179) and at the time of arthritis manifestation in patients who progressed to clinical arthritis (n=25). IL-40 was also analysed in the serum of age and sex-matched healthy controls (n=60). <i>In vitro</i> experiments were performed on PBMCs from at-risk individuals (n=10). PBMCs were exposed to recombinant IL-40 (10, 50, 100, and 250 ng/ml) and LPS. For inhibition studies, cells were pretreated with NFkB inhibitor prior to incubation with IL-40. Levels of IL-40 and IL-6 were measured by commercially available ELISA kits. <h3>Results</h3> Serum IL-40 was significantly up-regulated in at-risk individuals compared to healthy individuals (4.8 ± 0.2 vs. 1.5 ± 0.09 ng/ml, p&lt;0.0001). Moreover, the levels of IL-40 were significantly higher in the serum of double-positive (ACPA<sup>+</sup>/rheumatoid factor, RF<sup>+</sup>) compared to double-negative at-risk individuals (6.4 ± 0.7 vs. 4.5 ± 0.5 ng/ml; p&lt;0.05). Out of 175 at-risk individuals, only 25 developed clinical arthritis (within a median of 8,17 months of follow-up); however, we have not found a significant difference in the levels of IL-40 at baseline, and at the time of arthritis manifestation. To evaluate the pro-inflammatory effect of systemically elevated IL-40 in at-risk individuals, their PBMCs were exposed to recombinant IL-40 <i>in vitro</i>. We showed that IL-40 strongly enhances the secretion of IL-6 by PBMCs in a dose-dependent manner when compared to unstimulated cells (10 ng/ml of IL-40: p&lt;0.05; 50, 100, 250 ng/ml of IL-40: p&lt;0.01). Furthermore, the application of NFkB inhibitor to the PBMCs prior to the exposure to IL-40 significantly reduced the secretion of IL-6 when compared to IL-40 treated cells without NFkB inhibition (p=0.004). <h3>Conclusion</h3> This is the first study to demonstrate the up-regulation of IL-40 in the serum of individuals at risk of RA. Moreover, we showed that IL-40 induces the pro-inflammatory response in PBMCs at-risk individuals via NFκB dependent pathway. <h3>Acknowledgements</h3> This work was supported by AZV-NU21-05-00276, AZV-NU22-05-00226, MHCR 023728, SVV 260 523, and BBMRI-CZ LM2018125. <h3>Disclosure of Interests</h3> None Declared.